Deacetylation and Internal Cleavage of Polypeptides for N-Terminal Sequence Analysis

  • Madalina T. Gheorghe
  • Tomas Bergman


Proteins with a blocked N-terminus are common. Frequently the modification involves an acetyl-, formyl- or pyroglutamyl-moiety coupled to the α-amino group and direct sequence analysis by Edman degradation is not possible. Several enzymatic and chemical methods to remove the blocking group have been suggested (cf. Tsunasawa and Hirano, 1993), but they often suffer from poor yields and a large extent of undesirable peptide bond cleavage. Acetylation represents the most frequent N-terminal modification and is found in alcohol dehydrogenases among many other proteins. To circumvent the conventional approach to sequence analysis of blocked proteins (i.e. proteolytic cleavage, HPLC of fragments and internal sequence analysis) we have tested direct chemical deacetylation using a mixture of trifluoroacetic acid and methanol (Gheorghe et al., 1995). In this manner, drawbacks as high protein consumption, long handling times and inaccessibility of the N-terminal fragment to Edman degradation, are avoided. The protocol has been applied to both a synthetic peptide corresponding to the N-terminal segment of horse liver alcohol dehydrogenase and to the intact protein.


Intact Protein Edman Degradation Cyanogen Bromide Pyroglutamic Acid Direct Sequence Analysis 
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  1. Bergman, T. and Jörnvall, H., 1987 Electroblotting of individual polypeptides from SDS/polyacrylamide gels for direct sequence analysis, Eur. J. Biochem. 169: 9.PubMedCrossRefGoogle Scholar
  2. Bergman, T., Agerberth, B. and Jörnvall, H., 1991 Direct analysis of peptides and amino acids from capillary electrophoresis, FEBS Lett. 283: 100.PubMedCrossRefGoogle Scholar
  3. Bergman, T., 1994 Internal amino acid sequences via in situ cyanogen bromide cleavage, J. Prot. Chem. 13: 456.Google Scholar
  4. Egyhazi, E., Stigare, J., Holst, M. and Pigon, A., 1991 Analysis of the structural relationship between the DNA-binding phosphoproteins pp42, pp43 and pp44 by in situ peptide mapping, Molecular Biology Reports 15: 65.PubMedCrossRefGoogle Scholar
  5. Gardell, S.J., Craik, C.S., Clauser, E., Goldsmith, E.J., Stewart, C.-B., Graf, M. and Rutter, W.J., 1988 Anovel rat carboxypeptidase, CPA2. Characterization, molecular cloning, and evolutionary implications on substrate specificity in the carboxypeptidase gene family, J. Biol. Chem. 263: 17828.PubMedGoogle Scholar
  6. Gheorghe, M.T., Lindh, I., Griffiths, W.J., Sjövall, J. and Bergman, T., 1995 Analytical approaches to alcohol dehydrogenase structures, in: Enzymology and Molecular Biology of Carbonyl Metabolism 5, Weiner, H., Holmes, R.S., and Wermuth, B., eds., Plenum Press, New York, pp 417–426.CrossRefGoogle Scholar
  7. Hermansen, L.F., Bergman, T., Jörnvall, H., Husby, G., Rankly, I. and Sletten, K., 1995 Purification and characterization of amyloid-related transthyretin associated with familial amyloidotic cardiomyopathy, Eur. J. Biochem., 227: 772.PubMedCrossRefGoogle Scholar
  8. Jörnvall, H., 1970 Horse liver alcohol dehydrogenase. The primary structure of the protein chain of the ethanol-active isoenzyme, Eur. J. Biochem. 16: 25.PubMedCrossRefGoogle Scholar
  9. Kaiser, R., Holmquist, B., Hempel, J., Vallee, B.L. and Jörnvall, H., 1988 Class III human liver alcohol dehydrogenase. A novel structural type equidistantly related to the class I and class II enzymes, Biochemistry 27: 1132.PubMedCrossRefGoogle Scholar
  10. Kanda, Y., Goodman, D.S., Canfield, R.E. and Morgan, F.J., 1974 The amino acid sequence of human plasma prealbumin, J. Biol. Chem. 249: 6796.PubMedGoogle Scholar
  11. Kent, S.B.H., 1988 Chemical synthesis of peptides and proteins, Ann. Rev. Biochem. 57: 957.PubMedCrossRefGoogle Scholar
  12. Matsudaira, P., 1987 Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes, J. Biol. Chem. 262: 10035.PubMedGoogle Scholar
  13. Oppezzo, O., Ventura, S., Bergman, T., Vendrell, J., Jörnvall, H. and Avilés, F.X., 1994 Procarboxypeptidase in rat pancreas. Overall characterization and comparison of the activation processes, Eur. J. Biochem. 222: 55.PubMedCrossRefGoogle Scholar
  14. Schuppe-Koistinen, I., Moldéus, P., Bergman, T. and Cotgreave, I.A., 1995 Reversible S-thiolation of endothelial cell actin accompanies a structural reorganisation of the cytoskeleton, Endothelium, submitted.Google Scholar
  15. Tsunasawa, S. and Hirano, H., 1993 Deblocking and subsequent microsequence analysis of N-terminally blocked proteins immobilized on PVDF membrane, in: Methods in Protein Sequence Analysis, Imahori, K. and Sakiyama, F., eds., Plenum Press, New York, pp 45–53.Google Scholar

Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • Madalina T. Gheorghe
    • 1
  • Tomas Bergman
    • 1
  1. 1.Department of Medical Biochemistry and BiophysicsKarolinska InstitutetStockholmSweden

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